PowerPlex Y23 Developmental Validation
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1 PowerPlex Y23 Developmental Validation Jonelle Thompson, R&D Scientist September 2012
2 PowerPlex Y23 System Presentation outline Benefits of Y-STR Analysis Background information on PowerPlex Y23 Developmental requirements of SWGDAM PowerPlex Y23 Developmental Validation Data Update on Y-STR Statistical Databases US Y-STR Database YHRD Database 2
3 The Benefits of Y-STR Analysis Forensic casework on sexual assault evidence Relatively high amount of female DNA Mixtures with DNA from multiple male donors Paternity testing Familial searching Missing persons investigations Migration and evolutionary studies Historical and genealogical research 3
4 Overview of the PowerPlex Y23 System Includes all of the loci from PowerPlex Y as well as AmpFlSTR Yfiler Includes six Y-STR loci that are not in any other commercially available kit One system capable of extracted and direct amplification applications Amplify male genomic DNA in an excess of female genomic DNA No amplification products for female genomic DNA Rapid thermal cycling Compatible with the Applied Biosystems 3130 and 3500 Genetic Analyzers 4
5 PowerPlex Y23 System Six new loci with high gene diversity Size (bp) DYS576 (0.82) DYS389I DYS448 DYS389II DYS19 DYS391 DYS481 (0.85) DYS549 (0.79) DYS533 (0.75) DYS438 DYS437 DYS570 (0.86) DYS635 DYS390 DYS439 DYS392 DYS643 (0.93) DYS393 DYS458 DYS385 a/b DYS456 Y-GATA-H4 5
6 Gene Diversity of STR Loci in Y-STR Systems Locus Gene Diversity PowerPlex Y AmpFlSTR Yfiler PowerPlex Y 23 DYS DYS DYS DYS385 a/b 0.83 DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS389II 0.62 Y GATA H DYS389I 0.56 DYS DYS
7 Value of Additional Loci NIST concordance study: 12 sets of two individuals matched at all 17 AmpFlSTR Yfiler loci: With the addition of the new six loci, 8 of 12 sets were distinguished from each other (only 4 sets of two individuals still match) Remaining sets appear to be related individuals based on mitochondrial DNA and autosomal testing NIST manuscript submitted UNT concordance study Yfiler had 708 unique profiles Additional 6 loci alone had 557 unique profiles All 23 loci together had 758 unique profiles C. Davis, et. Al. Prototype PowerPlex Y23 System: A concordance study. Forensic Sci. Int. Genet. (2012). 7
8 PowerPlex Y23 System Thermal Cycling PowerPlex Y23 System 1 cycle 96 C for 2 min 30 or 26 cycles 94 C for 10 sec 61 C for 1 min 72 C for 30 sec 1 cycle 60 C for 20 min Extracted DNA 30 cycles 1 h 40 min total time Punches and swab extract 26 cycles 1 h 30 min total time AmpFlSTR Y-Filer 1 cycle 95 C for 11 min 27 or 30 cycles 94 C for 1 min 61 C for 1 min 72 C for 1 min 1 cycle 60 C for 80 min Extracted DNA 30 cycles >3 h total time Washed FTA 27 cycles
9 PowerPlex Y23 System Kit components Optimized for up to 17.5μl extracted DNA sample in 25μl reaction volume Component Pre-amplification 200 reactions (DC2320) 50 reactions (DC2305) PowerPlex Y23 5X Master Mix 4 x 250μl 1 x 250μl PowerPlex Y23 10X Primer Pair Mix 4 x 125μl 1 x 125μl 2800M Control DNA, 10ng/μl 25μl 25μl Water, Amplification Grade 5 x 1,250μl 5 x 1,250μl Post-amplification PowerPlex Y23 Allelic Ladder Mix 4 x 25μl 1 x 25μl CC5 Internal Lane Standard 500 Y23* 2 x 300μl 1 x 300μl * Optimized for PowerPlex Y23 only 9
10 PowerPlex Y23 Developmental Validation Data
11 SWGDAM Guidelines for Developmental Validation 2.1 Inheritance, mapping, detection, polymorphism 2.2 Species Specificity 2.3 Sensitivity studies 2.4 Stability 2.5 Reproducibility 2.6 Case-type samples/non-probative samples 2.7 Population studies 2.8 Mixture studies 2.9 Precision and accuracy 2.10 Specificity and robustness, balance and stochastic, known artifacts, positive and negative controls 11
12 PowerPlex Y23 System Developmental Validation Collaborators Four US laboratories have been identified to participate in the developmental validation Illinois State Police -sponsor for NDIS approval Sorenson Forensics Texas Department of Safety Casework DNA Section and CODIS Section Promega 12
13 2.1 Inheritance: Vermeulen, M. et al. (2009) Improving global and regional resolution of male lineage differentiation by simple single-copy Y-chromosomal short tandem repeat polymorphisms. For. Sci. Int. Genet. 3, DYS576 and DYS570 have mutation rates of >1%, considered rapidly mutating NIST studies for father son data in progress Mapping Hanson, E.K and J. Ballantyne. Comprehensive annotated STR physical map of human Y chromosome: Forensic implications. Legal Medicine 8 (2006) Article describes the six new loci included in PowerPlex Y23 13
14 2.2 Species specificity Promega tested on both the 3130 and 3500 Three higher primates: Chimpanzee, Gorilla, Orangutan Nine domestic animals: pig, horse, deer, cat, rabbit, chicken, dog, cow, mouse Seven microorganisms: E.coli, E.faecalis, S.servisiae, F. nucleatum, M. luteus, S. salivarius, S. mitis Results showed no artifacts with 10ng of domestic animal or microorganism DNA Partial profiles obtained from 500pg DNA from higher primates, however profile pattern was not consistent with human male profile ISP tested 4 primates: Gibbon, Macaque, DeBrazza s Monkey, Lemur 4 mammals: Cat, Dog, Fox, Mouse 2 microorganisms: C. perfringens and B.subtilis One allele was seen in the Macaque sample No other artifacts were seen from the domestic animals or microorganisms 14
15 2.2 Species Specificity 500pg Chimpanzee DNA 15
16 Developmental Validation 2.3 Sensitivity studies Extracted DNA 2 male DNA samples were tested by 3 laboratories All samples were tested in triplicate 1ng, 500pg, 250pg, 125pg, 62.5pg, 31.25pg Direct Blood on FTA Blood samples from 5 males were tested by 1 laboratory All samples tested in triplicate 1, 2, or 3 1.2mm punches Direct Buccal on FTA Buccal samples from 5 males were tested by 1 laboratory All samples tested in triplicate 1, 2, or 3 1.2mm punches 16
17 Percent of Loci Called Sensitivity of Extracted DNA 100% 90% 80% 70% 60% 50% 40% Male 1 Male 2 30% 20% 10% 0% 1ng 500pg 250pg 125pg 62.5pg 31.25pg Template Amount (pg)
18 2.3 Sensitivity Studies Extracted DNA full profiles consistently down to 62.5pg 500pg 250pg 125pg 62.5pg 31.25pg
19 Percent Loci Called 2.3 Sensitivity Studies Extracted DNA on 3500 Genetic Analyzer 100% 90% 80% 70% 60% 50% 40% Male 1 Male 2 30% 20% 10% 0% 1 ng 500 pg 250 pg 125 pg 62.5 pg pg DNA Template
20 2.3 Sensitivity Studies Extracted DNA on 3500 Genetic Analyzer 500pg 250pg 125pg 62.5pg 31.25pg
21 Percent of Loci Called 2.3 Sensitivity Studies Direct amplification of blood on FTA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1 punch 2 punch 3 punch Number of 1.2mm punches
22 2.3 Sensitivity Studies Direct amplification of blood on FTA 1 1.2mm punch 2 1.2mm punches 3 1.2mm punches
23 Percent of Loci Called 2.3 Sensitivity Studies Direct amplification of buccal on FTA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1 punch 2 punch 3 punch Number of 1.2mm punches
24 2.3 Sensitivity Studies Direct amplification of buccal on FTA 1 1.2mm punch 2 1.2mm punches 3 1.2mm punches
25 Developmental Validation 2.4 Stability studies inhibitors Three inhibitors Hematin: 500, 750, and 1000µM Humic acid: 100, 200, and 300ng/µl Tannic acid: 100, 200, and 300ng/µl 500ng extracted DNA from 2 males were tested in triplicate amplifications for each sample type Testing performed at 2 laboratories 25
26 1000uM 300ng/ul 300ng/ul 750uM, 200ng/ul 200ng/ul 500uM 100ng/ul 100ng/ul 2.4 Stability Studies Inhibitors 100% 90% 80% 70% Hematin 60% 50% Humic Acid 40% 30% Tannic Acid 20% 10% 0%
27 2.4 Stability Studies Inhibitors Hematin No Inhibitor 500uM 750uM 1000uM
28 2.4 Stability Studies Inhibitors Humic acid No Inhibitor 100ng/µl 200ng/µl 300ng/µl
29 2.4 Stability Studies Inhibitors Tannic acid No Inhibitor 100ng/µl 200ng/µl 300ng/µl
30 Developmental Validation 2.5 Reproducibility Extracted DNA NIST 2395 Tested by 3 laboratories Blood on FTA 1 1.2mm punch 2 males Tested by 3 laboratories Buccal on FTA 2 1.2mm punch 2 males Tested by 2 laboratories 30
31 2.5 Reproducibility Extracted DNA: NIST 2395 NIST A NIST B NIST C NIST D NIST E DYS DYS389 I DYS DYS389 II DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS DYS385 12,15 14,17 17,20 14,15 13,15 DYS YGATAH Note, samples were run on both the 3130 and All samples were concordant with certificate from NIST for all loci available, and were consistent across all amplifications and platforms for the ones not available.
32 2.5 Reproducibility Blood and buccal on FTA Promega Sorenson Forensics Texas DPS Sample Type Blood Blood Blood # Reps Male # Reps Male Concordant? YES YES YES Sample Type Buccal Buccal # Reps Male # Reps Male Concordant? YES YES
33 2.6 Case-type samples/non-probative samples Case samples tested Sperm and epithelial fractions from: Underwear Bedding Coat Shirt Epithelial fractions from: Underwear Blue jeans Bloodstain: Sheet 33
34 2.6 Case-type samples/non-probative samples This sample is actually an unstained area adjacent to an area where semen was identified. Autosomal STR results identified a complete female source profile associated with the person wearing the garment (underwear) and 4 minor alleles plus Amelogenin Y. 34
35 2.7 Population Study -Concordance University of North Texas Health Science Center > 950 US population samples tested C. Davis, et. Al. Prototype PowerPlex Y23 System: A concordance study. Forensic Sci. Int. Genet. (2012). Concordance in 17 common loci with AmpFISTR Yfiler kit Null allele in DYS448 with duplication in DYS576 due to a 42 base deletion. This deletion is also observed with AmpFlSTR Yfiler and has been published Budowle, et. al., (2008) Null allele sequence structure at the DYS448 locus and implications for profile interpretation, Int J Legal Med 122: National Institute of Standards and Technology > 600 US population samples tested Concordance in 17 common loci with AmpFlSTR Yfiler kit and 4 new loci (NIST primer sequences) 35
36 2.8 Mixture studies Male/Male Mixtures 2 male/male mixture sets sent to laboratories, 500pg total DNA 3 laboratories completed studies Samples amplified in triplicate 19:1, 9:1, 3:1, 1:1, 1:3, 1:9, and 1:19 Male/Female Mixtures Male Varied/Female constant 2 male/female mixture sets sent to each laboratory 3 laboratories completed studies Samples amplified in triplicate Female held constant at 400ng; male at 500pg, 250pg, 125pg, 62.5pg, 31.25pg Male Constant/Female Varied 1 male mixture set used 1 laboratory completed study Samples amplified in triplicate Male held constant at 125pg or 500pg; female at 500ng, 1000ng, 2000ng, or 3000ng 36
37 Percentage of Unique Minor Profile Detected 2.8 Mixture Studies Male/Male Mixtures 100% 90% 80% 70% 60% 50% 40% M1M2 M2M3 30% 20% 10% 0% 19:1 9:1 3:1 1:1 1:3 1:9 1:19 Mixture Ratio
38 2.8 Mixture Studies Male/Male Mixtures, 19 to 1 ratio
39 2.8 Mixture Studies Male/Male Mixtures, 1 to 1 ratio
40 Percentage of Loci Called 2.8 Mixture Studies Male/female Mixtures decreasing male DNA with 400ng female DNA 100% 90% 80% 70% 60% 50% 40% Male 1 Male 2 30% 20% 10% 0% 500pg 250pg 125pg 62.5pg 31.25pg Template Amount (pg)
41 2.8 Mixture Studies Male Varied/Female Constant Mixtures 62.5pg of male DNA with 400ng female DNA (6400X)
42 2.8 Mixture Studies Male Constant/Female Varied Mixtures 125pg male DNA with 3000ng of female DNA (24,000X)
43 2.9 Precision study Precision was performed at each laboratory Both 3130 and 3500 series of Genetic analyzers were tested 43
44 Standard Deviation 2.9 Precision Studies AB 3130 Precision of 14 Ladders on 3130 with POP Fragment Size (bp) 44
45 Standard Deviation 2.9 Precision Studies AB 3500xl Precision of 8 Ladders on 3500xl with POP Fragment Size (bp) 45
46 Specificity and robustness Reaction Volume - 25µl vs 12.5µl Extracted DNA - data not yet available Blood on FTA 1 1.2mm punch Buccal on FTA 1 or 2 1.2mm punch Cycle Number Extracted DNA 28, 30, and 32 cycles Blood on FTA, 1 1.2mm punch 25, 26, and 27 cycles Buccal on FTA, 2 1.2mm punch 26, 27, and 28 cycles Bode Buccal Collector, 1 1.2mm punch, with PunchSolution - 25, 26, and 27 cycles Blood on S&S 903, 1 1.2mm punch with PunchSolution - 25, 26, and 27 cycles Omni swabs with SwabSolution, 1 entire swab head- 25, 26, and 27 cycles Cotton swabs with SwabSolution, 1 entire swab head - 25, 26, and 27 cycles 46
47 Specificity and robustness Annealing Temperature - 59 C, 61 C, 63 C, Extracted DNA 500pg male DNA Blood on FTA 1 1.2mm punch Buccal on FTA 2 1.2mm punch PCR Reaction Components, magnesium and EDTA titration Extracted DNA 500pg male DNA Primer Concentration 0.75X, 1X, 1.25X Extracted DNA 500pg male DNA data not yet available Blood on FTA 1 1.2mm punch Buccal on FTA 2-1.2mm punch 47
48 % of Loci Called Specificity and robustness Reaction Volume Blood on FTA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 25µl reaction 12.5µl reaction Reaction Volume
49 Specificity and robustness Reaction Volume Blood on FTA 1-1.2mm punch - 25µl 1-1.2mm punch 12.5µl
50 Specificity and robustness Reaction Volume Buccal on FTA 100% 90% 80% 70% 60% 50% 40% 1-1.2mm punch 2-1.2mm punch 30% 20% 10% 0% 25µl reaction, Buccal 12.5µl reaction, Buccal
51 Specificity and robustness Reaction Volume Buccal on FTA, 1 1.2mm punch 25µl 12.5µl
52 % of Loci Called Specificity and robustness Cycle Number Extracted DNA 100% 90% 80% 70% 60% 50% 40% 28 cycles 30 cycles 32 cycles 30% 20% 10% 0% 1 ng 500 pg 250 pg 125 pg 62.5 pg pg Template Amount
53 Specificity and robustness Cycle Number 62.5pg, 28 cycles
54 Specificity and robustness Cycle Number 62.5pg, 30 cycles
55 Specificity and robustness Cycle Number 62.5pg, 32 cycles
56 Specificity and robustness Cycle Number Direct Amplifications 100% 90% 80% 70% 60% 50% 40% 1 fewer cycle than recommended Recommended Cycle Number 1 more cycle than recommended 30% 20% 10% 0% Bode Buccal Omni Swabs Blood on S&S903 Buccal on FTA Blood on FTA
57 Specificity and robustness Annealing Temperature Extracted DNA, 100pg data
58 Specificity and robustness Annealing Temperature Extracted DNA 59 C 61 C 63 C
59 Percent of Loci Called Specificity and robustness Annealing Temperature Blood on FTA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Blood, 59 C Blood, 61 C Blood, 63 C Annealing Temperature
60 Specificity and robustness Annealing Temperature Blood on FTA 59 C 61 C 63 C
61 Percent of Loci Called Specificity and robustness Annealing Temperature Buccal on FTA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Buccal, 59 C Buccal, 61 C Buccal, 63 C Annealing Temperature
62 Specificity and robustness Annealing Temperature Buccal on FTA 59 C 61 C 63 C
63 Percent Loci Called Specificity and robustness PCR reaction components Extracted DNA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1.25X MgCl2 1X MgCl2 0.1mM EDTA 0.25mM EDTA 0.5mM EDTA 1mM EDTA Magnesium Chloride and EDTA Concentration
64 Specificity and robustness PCR reaction components Extracted DNA 1.25X MgCl 2 1X MgCl 2 0.1mM EDTA 0.25mM EDTA 0.5mM EDTA 1mM EDTA
65 Percent Loci Called Specificity and robustness Primer Concentration Blood on FTA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Blood, 0.75X Primer Blood, 1X Primer Blood, 1.25X Primer
66 Specificity and robustness Primer Concentration Blood on FTA 0.75X 1X 1.25X
67 Percent Loci Called Specificity and robustness Primer Concentration Buccal on FTA 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Buccal, 0.75X Primer Buccal, 1X Primer Buccal, 1.25X Primer
68 Specificity and robustness Primer Concentration Buccal on FTA 0.75X 1X 1.25X
69 Balance and stochastic 500pg Male DNA 69
70 Balance and stochastic 62.5pg Male DNA
71 Known Artifacts Artifacts Listed in the Technical Manual 71
72 Known Artifacts Artifacts Listed in the Technical Manual POP7 The PowerPlex Y23 System is optimized for POP-4 polymer. We recognize that some laboratories use POP-7 polymer and therefore have included a protocol in this manual. Some DNA-independent artifacts migrate in the panel range with the POP-7 polymer. Global filters used for database analysis will generally filter these artifact peaks. However, these peaks may be labeled with casework samples. Internal validation should be performed and interpretation guidelines created that describe the artifacts and their impact on data analysis. 72
73 Positive Control 500pg 2800M 73
74 Negative Control
75 Stutter Samples
76 PowerPlex Y23 System Stutter Values 3500 minus stutter 3500 plus stutter Average Occurrence Average Occurrence n=105 Average St.Dev + 3SD Max Count % n=105 Average St.Dev + 3SD Max Count % DYS % DYS % DYS389I % DYS389I % DYS % DYS % DYS389II % DYS389II % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % DYS % Y-GATA % Y-GATA % 3500 DYS19 N DYS19 N+2 Average Occurrence Average Occurrence N=105 Average St.Dev + 3 STD Max Count % N=105 Average St.Dev + 3 STD Max Count % N % N % 76
77 PowerPlex Y23 System Stutter Filters for GeneMapper ID-X Marker Name DYS576 Marker Name DYS Minus Minus Marker Name DYS389 I Marker Name DYS Minus Minus Marker Name DYS448 Marker Name DYS Minus Minus Marker Name DYS389 II Marker Name DYS Minus Minus Marker Name DYS19 Marker Name DYS Minus Minus Minus Plus Plus Marker Name DYS643 Marker Name DYS Minus Minus Marker Name DYS393 Marker Name DYS Minus Minus Marker Name DYS Plus Minus Marker Name DYS549 Marker Name DYS Minus Minus Marker Name DYS533 Marker Name DYS Minus Minus Marker Name DYS438 Marker Name YGATAH Minus Minus Marker Name DYS Minus 77
78 PowerPlex Y23 System Stutter Filters for GeneMapper ID The value of 0.06 will filter the plus stutter for DYS481 (5.5%) but not for DYS392 (10.5%). This is noted in the Technical Manual Minus stutter is included in the panels file #GeneMapper ID v3.2 Version GM v 3.0 Kit type: MICROSATELLITE Chemistry PowerPlex null Panel PowerPlex null DYS576 blue DYS389 I blue DYS448 blue DYS389 II blue DYS19 blue DYS391 green DYS481 green DYS549 green DYS533 green DYS438 green DYS437 green DYS570 yellow DYS635 yellow DYS390 yellow DYS439 yellow DYS392 yellow DYS643 yellow DYS393 red DYS458 red DYS385 red , DYS456 red YGATAH4 red
79 Y-STR Statistical Databases
80 US Y-STR Database
81 US Y-STR Database As of 8/9/12, 1375 samples contained all 23 loci. This number is anticipated to increase with submissions in the upcoming months. 81
82 Updated Y-HRD Database Interface Confidential and Proprietary. Not for Further Disclosure.
83 Summary
84 Performance Summary PowerPlex Y23 System is a very flexible system that is capable of working for multiple applications including casework (extracted) samples as well as multiple substrates for direct amplification Full profiles obtained consistently at 62.5pg of male DNA Ability to overcome excessive amounts of female with full profiles of 125 pg in 3000 ng of female DNA Rapid cycling of 1 hour 40 minutes for casework samples and 1 hour 30 minutes for direct amplification samples Robust against many of the commonly encountered inhibitors; humic acid, hematin, and tannic acid 84
85 Supported instruments & software summary Thermal Cycler: GeneAmp PCR System 9700 (Applied Biosystems) Capillary Electrophoresis Instrument: 3500 & 3500xL Genetic Analyzer (Applied Biosystems) 3130 & 3130xl Genetic Analyzer (Applied Biosystems) ABI PRISM 3100 & 3100-Avant Genetic Analyzer (Applied Biosystems) Data Analysis Software: GeneMapper ID-X Software, version 1.2 (Applied Biosystems) Includes plus stutter filters for DYS481 and DYS392 (trinucleotide repeat loci) Includes N-2 and N+2 filters for DYS19 GeneMapper ID Software, version 3.2 (Applied Biosystems) 85
86 Acknowledgements Developmental Collaborators Illinois State Police Sorenson Forensics Texas DPS DNA section CODIS section Population Data Collaborators NIST UNT R&D Doug Storts Cindy Sprecher Jonelle Thompson Margaret Ewing Dawn Rabbach Patricia Fulmer Manufacturing Kris Pearson Katie Hebble Anna Markhardt Kara Raymond Jennifer Setlak Charlie Stollberg Oligo Synthesis QA Ned Reimer Kevin DeGrand Alyssa TenHarmsel Marketing Lotte Downey Ann MacPhetridge Rohaizah James Melissa Schwandt Project Manager Brian McNamara 86
87 Questions?
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